1. Field of the Invention
This invention resides in the field of affinity chromatography, and addresses concerns arising from the labile character of ligands coupled to a support as the stationary phase.
2. Description of the Prior Art
Affinity chromatography is widely used for separating and detecting components in biological samples and for the isolation or purification of biological species or recombinant species from clinical samples, from cell growth cultures, or from any medium in which the species are produced or can be extracted. Affinity chromatography is commonly performed by passing a liquid medium containing the species of interest through a column or membrane to which a ligand is bound as a stationary phase, the ligand being one to which the species of interest binds by an affinity-type interaction. Affinity chromatography that is used to isolate and purify species is also termed “affinity extraction,” and the species-ligand interaction in this type of extraction is one that occurs with sufficient specificity to differentiate between the species of interest and other species in the source liquid. Affinity extraction techniques include immunoextraction in which the ligands are antibodies; protein-protein extractions using such ligands as wheat germ agglutinin, concanavalin A, protein A, and protein G; and interactions involving non-protein species such as heparin or nucleic acids. Once immobilized on the solid phase by the affinity interaction, the species of interest is removed from the solid phase by an appropriate change in conditions such as a change in pH or the introduction of a detergent, chaotrope, salt, competitive binding species, or any agent that will overcome or lessen the binding affinity of the species to the solid phase. The types of changes that will be effective in releasing the bound species in particular systems are well known in the art of affinity chromatography.
The ligand is typically a protein or other affinity-binding species that is coupled to a solid support by covalent bonds, the support often having been activated to facilitate the formation of such bonds. Activation commonly involves the placement of a reactive group, such as for example an epoxide group, on the support surface. The linkage between the ligand and the support is labile, however, often resulting in dissociation of the ligand from the support during the passage of liquids through the medium. In addition, leaching can occur by enzymatic or chemical degradation of the ligand itself, such as proteolysis of protein affinity ligands by proteases in the process stream, cleavage of nucleic acids by endo- and exo-nucleases in the process stream, etc. The affinity ligand that is leached from the affinity medium as a result of this dissociation may be small compared to the amount of ligand remaining on the support, but even a small amount of leached ligand can seriously contaminate the otherwise purified species eluted from the medium. When a therapeutic agent that is either biologically derived or produced by recombinant chemistry is contaminated with a leached affinity ligand, the leached ligand can recombine with the agent and thereby impede the effectiveness of the agent, or combine with, or impede the functions of, other species such as membranes, cell walls, or enzymes in the patient's body to cause harm. Concavalin A, for example, is an affinity ligand that is used for purifying lysosomal enzyme preparations, but is known to leach from affinity columns and contaminate the enzyme preparations, particularly by activating T cells in the patient to whom the enzyme preparation is administered. To eliminate these types of contamination, the leached ligands must be removed, and this is typically performed by separations downstream of the affinity column or membrane. This adds cost and time to the preparation.